Linear B-cell epitopes are critically important for immunological applications, such as vaccine design, immunodiagnostic test, and antibody production, as well as disease diagnosis and therapy. The accurate identification of linear B-cell epitopes remains challenging despite several decades of research. In this work, we have developed a novel predictor, Identification of Linear B-cell Epitope (iLBE), by integrating evolutionary and sequence-based features. The successive feature vectors were optimized by a Wilcoxon-rank sum test. Then the random forest (RF) algorithm using the optimal consecutive feature vectors was applied to predict linear B-cell epitopes. We combined the RF scores by the logistic regression to enhance the prediction accuracy. iLBE yielded an area under curve score of 0.809 on the training dataset and outperformed other prediction models on a comprehensive independent dataset. iLBE is a powerful computational tool to identify the linear B-cell epitopes and would help to develop penetrating diagnostic tests. A web application with curated datasets for iLBE is freely accessible at http://kurata14.bio.kyutech.ac.jp/iLBE/. B-cell epitopes (BCEs) are specific regions of immunoglobulin molecules that can stimulate the immune system, which contributes to diagnostic test, antibody production, and vaccine design BCEs are categorized into two groups: continuous and discontinuous ones In this work, we have established a computational, global predictor named Identification of Linear B-cell Epitope (iLBE) by integrating sequence and evolutionary features. For evolutionary features, we considered the position-specific scoring matrix (PSSM) and composition of profile-based amino acids frequency (PKAF) encoding descriptors. For primary sequence features, we considered amino-acid index property (AIP) and amino acid frequency composition (AFC). To optimize the consecutive feature vectors, a non-parametric Wilcoxon-rank sum (WR) test was employed. Then the random forest (RF) algorithm using the optimal consecutive feature vectors was used to identify linear BCEs. By the combination of the RF scores through logistic regression (LR), the iLBE yielded better performance than other predictors. Finally we implemented iLBE as a user-friendly web application. The computational outline of the iLBE is shown in Experimentally well-characterized datasets of BCEs are needed to develop an accurate machine learning (ML) classifier. We pulled an experimental dataset of linear peptides from the Immune Epitope Database (IEDB), which consists of the verified positive samples (BCEs) and negative samples (non-BCEs) The PSSM profile was generated using the PSI-BLAST (a version of 2.2.26+) with the whole Swiss-Prot non-redundant-protein database (a version of December 2010). We used two onset parameters: an iteration times of 3 and E-value cutoff of 0.0001 m and m + k +1, the composition scores were measured or standardized by the following formula:W is the peptide length of BCEs, a k-spaced residues characterized as i{k}xjx  represent 20 types of common residues, and T means that ix{k}jx performs T times for the positive /negative samples. PSSM  signifies the score of amino acid ix at mth row in ix{k}jx, and PSSM  indicates the score of residue jx at the row of (m + k + 1)th. An optimum value of k is 0 or 1, and the dimension of PKAF is 800.After generating the PSSM profile, we generated PKAF feature vectors In addition, we employed a similarity-search-based tool of BLAST (version of ncbi-blast-2.2.25+) to examine whether a query peptide belongs to BCEs or not W, a (W × 8) dimensional vector was generated via the AIP encoding.The AIP database (a version of 9.1) contained numerical indices of biochemical and physicochemical properties of amino acids 400 types of residue pairs. An optimal value of k, which signifies the frequency of any two-amino acid pairs, was set to 0 or 1. Consequently, 20 × (k + 1) × 20 = 800 distinguished residue pairs were generated. The feature vector was then calculated and standardized by the following formula:Ntotal is the length of epitope in the total composition residues. If epitope length W is 24 and k is 0 or 1, then tNotal = W − k − 1 is 23 or 22, respectively.  represents the frequency vector of amino acid pairs within the BCEs and non-BCEs.The AFC encoding is widely used for representing short sequence peptide motifs Uncorrelated and redundant features may exist in the generated feature vectors, which can affect the accuracy of a prediction model 1(S), H2(S), …, HN(S)}, which are built on N random subcategories of the training samples. This forest was trained with the bagging method to build an ensemble of decision trees. The general idea of the bagging method is that learning models are assembled to increase the global performance. Details in the RF algorithm were provided in previous studies https://cran.r-project.org/web/packages/randomForest/).To construct a prediction model, an RF classifier was used. It is a supervised ML algorithm and widely used in bioinformatics research https://www.csie.ntu.edu.tw/~cjlin/libsvm/) was used for the SVM algorithmThree commonly used ML algorithms, naive Bayes (NB) nβ is the regression coefficient, nR is the RF score of each feature, and α is the regression constant. The R software package (https://cran.r-project.org/) was employed for a generalized model of LR.To construct the final model of iLBE, the respective RF scores evaluated from the four features  were combined using a LR algorithm. The LR algorithm was effectively used in ubiquitination site prediction vs. 1 − Sp) and measured the area under curve (AUC) values To examine the performance of iLBE, four widely-used statistical measures, represented as sensitivity (Sn), specificity (Sp), accuracy (Ac), and Matthews correlation coefficient (MCC), were defined as:The prediction performance was assessed using 10-fold cross-validation (CV) test on the training model until no further improvement occurred after each round of optimization parameters. The training dataset was separated into 10 groups, where 9 of the groups were used for training and the remaining one for test. This selection process was repeated 10 times to assess the average performance of the 10 models.To develop the prediction model, we first compiled the training and independent datasets in the same manner as described by Manavalan et al. [28] (see Dataset preparation section). The prediction result was evaluated based on the criterion of whether the indication measure  exceeds a threshold value. The AUC value of the ROC curve was evaluated, with the threshold value of the RF score changed to classify a BCE or non-BCE. The threshold value determines the desirable balance to successfully detect positive and negative BCEs. The true positive rate (Sn) and the false positive rate (1 − Sp) were calculated for each threshold value of the RF scores. The high-, moderate-, and low-level thresholds were determined based on RF scores of 0.485, 0.410, and 0.360, respectively, which corresponded to Sp levels of 0.866, 0.747, and 0.636 in the training set results, respectively.http://kurata14.bio.kyutech.ac.jp/iLBE/. The web application was written in various programming languages including Perl, R, CGI scripts, HTML, and PHP. The server takes antigen epitopes written with 20 types of common amino acids in the FASTA format. When the submission job is completed, the server returns the prediction results with a combined RF score of the predicted BCEs in a tabular form to the output webpage with the job ID and a query peptide. Users can save the ID for a future query and the iLBE server stores this ID for a month.To provide a prediction service of potential BCEs to the scientific community, an accessible web page of the iLBE was established at t-test with P < 0.05  pair of residues SP is important and enriched in BCEs .We found that the AFC scheme presented the highest AUC, Sn, Ac, and MCC for all four single encoding methods . To inveTo optimize the length of short epitopes, we investigated the different lengths  of BCEs using the four encoding schemes of AIP, PSSM, AFC, and PKAF and their combined scheme (iLBE) . The RF The RF algorithm was characterized in comparison with the widely-used ML algorithms of NB, SVM, and ANN on the same training dataset. AUC values of predictions using the four algorithms without any feature selection were evaluated by a 10-fold CV test. As shown in P < 0.05, paired two sample t-test).We evaluated the prediction performance of the proposed iLBE with existing approaches on the same dataset. First, we employed the training dataset to compare the performance of iLBE with those of the LBtope and iBCE-EL models, which are the state-of-the-art predictors and publicly accessible. As shown in (To investigate the effects of combination methods on the prediction performance, we built a competitive model of iLBE, which arranges the four encoding vectors of AFC, AIP, PSSM, and PKAF in a row, instead of the use of LR. It is named as the sequential combination model. The resultant total dimension was 2192. The top 380 feature vectors were collected and rearranged in the ascending order of WR values. The WR-optimized feature vectors were used to train the RF classifier via a 10-fold CV test. The sequential combination model with and without feature collection approaches yielded AUC values of 0.778 and 0.767 on the training dataset, respectively A, and prB. The LR and was We have developed a novel computational predictor, iLBE, which accurately predicts BCEs for both the training and independent datasets. iLBE outperformed existing state-of-the-art predictors LBtope and iBCE-EL. The iLBE model combined the sequence-based features and evolutionary information, while the LBtope and iBCE-EL predictors only used sequence-based encoding methods. iLBE employed the LR-based combined model of the RF-based classifiers, while LBtope and iBCE-EL used SVM and an ensemble ML model, respectively. Importantly, iLBE allows the use of various threshold values at high, moderate, and low levels to demonstrate whether a BCE is highly positive or negative, which is not available in the existing prediction tools. As a complementary to the experimental strategies, iLBE provides insight into the functional and significant characteristics of BCEs. A user-friendly web-application was also developed for easy use by the immunological research community.http://kurata14.bio.kyutech.ac.jp/iLBE/.A web application with curated datasets for iLBE is freely accessible at Md. Mehedi Hasan: Conceptualization, Data curation, Methodology, Formal analysis, Software, Writing - original draft. Mst. Shamima Khatun: Data curation, Formal analysis, Methodology, Software. Hiroyuki Kurata: Conceptualization, Supervision, Writing - original draft. All authors read and approved the final manuscript.The authors have declared no competing interests.


Thromboelastometric parameters were significantly different between groups . More patients in the high-IL-6 group received MT (p <0.0001). The ROCs revealed an area under the curve of 0.76 vs. 0.82 for the high-IL-6 group for receiving MT in the first 6 and 24 h. IL-6 ≥ 350 pg/mL predicted MT within 6 and 24 h with a sensitivity of 45% and 58%, respectively, and a specificity of 89%. IL-6 ≥ 350 pg/mL appears to be a reasonable early predictor for coagulopathy and MT within the first 6 and 24 h intervals. Large-scale prospective studies are warranted to confirm these findings.Severe bleeding remains a prominent cause of early in-hospital mortality in major trauma patients. Thus, prompt prediction of patients at risk of massive transfusion (MT) is crucial. We investigated the ability of the inflammatory marker interleukin (IL)-6 to forecast MT in severely injured trauma patients. IL-6 plasma levels were measured upon admission. Receiver operating characteristic curves (ROCs) were calculated, and sensitivity and specificity were determined. In this retrospective study, a total of 468 predominantly male (77.8%) patients, with a median injury severity score (ISS) of 25 (17–34), were included. The Youden index for the prediction of MT within 6 and 24 h was 351 pg/mL. Patients were dichotomized into two groups: (i) low-IL-6 < 350 pg/mL and (ii) high-IL-6 ≥ 350 pg/mL. IL-6 ≥ 350 pg/mL was associated with a lower prothrombin time index, a higher activated partial thromboplastin time, and a lower fibrinogen concentration compared with IL-6 < 350 pg/mL ( Exsanguination remains a leading but potentially preventable cause of early in-hospital mortality in severely injured patients . ApproxiIn addition to the quick detection of the underlying coagulopathy, the rapid estimation of the risk of massive transfusion (MT) is warranted. A variety of MT prediction scores have been developed so far ,6,7,8. IAlthough intensive crosstalk between inflammation and coagulation has been well-described for sepsis, less is known about the impact of IL-6 on coagulopathy and transfusion requirements in major trauma ,19. DataThe primary objective of this retrospective study was to evaluate the association between the circulating IL-6 and blood transfusion requirements. We specifically hypothesized that in trauma patients, IL-6 blood concentration measured upon ER admission is associated with coagulopathy and predicts the need for an MT.Following the local ethics committee approval (AUVA EK 08/2020), we performed a retrospective analysis of trauma patients admitted between January 2012 and December 2018 to the AUVA Trauma Center Salzburg, Austria, a certified level 1 Trauma Hospital. All patients >17 years in whom IL-6 and rotational thromboelastometry (ROTEM) measurements were performed upon ER admission were eligible for analysis. Exclusion criteria were patients <17 years, patients missing IL-6 and ROTEM data, patients transferred from other hospitals and transferred from our ER in other facilities, and patients in whom further therapy was withheld due to futility. A flow chart outlines included and excluded patients .® (TR-DGU) of the German Trauma Society . This documentation was used for analysis, and missing variables were extracted from the anesthesia and intensive care unit (ICU) database (COPRA 6) and the electronic patient information system (ASTRA).Since 2012, prospective, standardized, and anonymous documentation of all major trauma patients admitted to the AUVA Trauma Centre has been established, and these data were forwarded to the central database of the TraumaRegister DGU® delta device ; from July 2016 onwards, a fully automated cartridge-based ROTEM® sigma device has been used. Moreover, arterial blood gas analyses were used to quantify base excess  and lactate concentration .Upon ER admission, the following laboratory measurements are performed in major trauma patients on a routine basis: full blood cell count with differential and standard coagulation parameters, including fibrinogen concentration ; prothrombin time index , activated partial thromboplastin time ; and ROTEM analysis. From January 2015 to June 2016, EXTEM, INTEM, and FIBTEM analyses were run on a ROTEMIL-6  measurement is also part of our standard laboratory panel upon ER admission of severely injured patients.®, COPRA System GmbH, Berlin, Germany) and a special database where all allogeneic blood transfusions have to be documented . Massive transfusion was defined as a red blood cell (RBC) transfusion ≥6 U within 6 h or ≥10 RBC/24 h [Coagulation therapy was primarily based on coagulation factor concentrates and guided by ROTEM test results . HemostaRBC/24 h .Distribution of the data was assessed using the Shapiro–Wilk test. Continuous variables are expressed as median and interquartile ranges . Categorical variables are reported as numbers and percentages (%), and compared between groups using Fisher’s exact or chi-squared tests. For analyzing the between-group differences in the metric variables, the Mann–Whitney U test was used. The sensitivity and specificity of the analyzed data were calculated by receiver operating characteristic (ROC) curves, and predictive capacity is expressed by the area under the curve (AUC) value. The optimal IL-6 level for prediction of MT at 6 and 24 h was determined by Youden’s index (sensitivity + specificity − 1)Multiple logistic regression models were fitted to the data to adjust for potential associations with other variables. In the first step, a list of several potential variables were specified. Subsequently, the coefficients/odds ratios were estimated, using an IL-6 value of ≥350 pg/mL as a cutoff for the dichotomization of the outcome, and a forward variable selection procedure was applied.p-values below 0.05 were considered significant [Statistical calculations were performed using GraphPad Prism  and R statistics (R Core Team 2019). nificant .A total of 468 predominantly (77.8%) male patients were included in the current study. The median age of the patients was 49 (33.3–64.0) years, the Injury Severity Score (ISS) was 25 (17–34), and the new ISS (NISS) was 29 (22–43). The median intensive care unit (ICU) length of stay was 9 (4–16) days. A total of 29 (6.2%) patients received MT and 50 (10.6%) died.The Youden’s index calculation for the prediction of massive transfusion within 6 and 24 h was 351 pg/mL. According to previous reports , we usedp < 0.001 for all). The abbreviated injury score (AIS) for most body regions was significantly higher in patients in the high-IL-6 group compared with the low-IL-6 group. Upon ER admission, patients in the high-IL-6 group were in a more severe shock state compared with the low-IL6 group, as indicated by significantly lower systolic blood pressure, a higher heart rate, and a lower hemoglobin concentration upon admission. This is also reflected by a lower base excess and a higher lactate concentration .Importantly, high-IL-6 patients were frequently more coagulopathic upon ER admission compared with low-IL-6 patients . Both stp < 0.05 for all; Coagulopathy was also evident, given that patients in the high-IL-6 group received significantly higher amounts of fibrinogen concentrate and prothrombin complex concentrate within the first 6 and 24 h compared with the low-IL-6 group  .To the best of our knowledge, this is the first study that investigated the association of IL-6 plasma concentration upon ER admission with coagulopathy and allogeneic blood transfusion requirements in a cohort of severely injured trauma patients. The current study revealed that IL-6 is a valid early prognostic marker for MT. IL-6 ≥ 350 pg/mL upon hospital admission predicted MT within 6 and 24 h with a sensitivity and specificity comparable to previously published MT prediction scores .Patients with severe trauma upregulate both pro- and anti-inflammatory cytokines  and IL-6The quantity of IL-6 released into the blood stream is primarily related to the extent of the tissue trauma and, to a lesser degree, to the severity and duration of shock ,26. PatiIn contrast with trauma, the impact of hemorrhagic shock alone on IL-6 release remains controversial. Roumen et al. observed that the occurrence of shock has only a minor impact on IL-6 generation . ConversPatients with an established TIC upon ER admission are prone to higher complication rates and increased mortality compared with patients without coagulopathy ,3. It isAn early prediction of MT upon ER admission is highly warranted, but remains challenging. Brockamp et al. validated six different MT prediction scores and reported only limited sensitivity and specificity . The higThere are several limitations in the current study, which must be considered when interpreting its findings. First, the analyses were performed retrospectively, and our study suffers from the inherent shortcomings of such a data evaluation.Second, IL-6 concentration in the blood is not only related to the severity of tissue trauma but also to a time-dependent variable. Thus, the time elapsed between the injury and hospital admission is a crucial factor as IL-6 typically increases over a short time period ; it takeThird, hemostatic therapy in our hospital is primarily based on coagulation factor concentrates. Small prospective and retrospective studies revealed that this strategy might result in a lower incidence in MT compared with a plasma-based coagulation therapy ,37,38. TForth, in contrast with point-of-care devices, such as viscoelastic and blood gas analyzers, IL-6 measurements are time consuming. Thus, results for decision making are available only with considerable time delay.High IL-6 level upon emergency room admission is associated with a substantial impairment of hemostasis. Trauma patients with plasma IL-6 exceeding 350 pg/mL are prone to MT. Sensitivity and specificity, as well as positive and negative predictive values, are comparable to the current MT prediction scores. Our findings support the use of IL-6 measurements upon ER admission as a fast laboratory parameter to identify coagulopathic trauma patients with the potential need for MT. However, large-scale studies are warranted to confirm our findings.


Both groups received 30 min of upper limb and hand movement and sit-to-stand training. After that, the I-Walk group received 30 min of I-Walk training, while the control followed a 30-minute overground training program. All the individuals were trained 3 days/week for 8 weeks. The primary outcome of the motor recovery of lower limb impairment was measured using the Fugl–Meyer Assessment (FMA). The secondary outcomes for gait performance were the 6-minute walk test (6 MWT), the 10-meter walk test (10 MWT), and the Timed Up and Go (TUG). The two-way mixed-model ANOVA with the Bonferroni test was used to compare means within and between groups. The post-intervention motor and sensory subscales of the FMA significantly increased compared to the baseline in both groups. Moreover, the 6 MWT and 10 MWT values also improved in both groups. In addition, the mean difference of TUG in the I-Walk was higher than the control. The efficiency of I-Walk training was comparable to overground training and might be applied for chronic stroke gait training in the community.Chronic stroke leads to the impairment of lower limb function and gait performance. After in-hospital rehabilitation, most individuals lack continuous gait training because of the limited number of physical therapists. This study aimed to evaluate the effects of a newly invented gait training machine  on lower limb function and gait performance in chronic stroke individuals. Thirty community-dwelling chronic stroke individuals were allocated to the I-Walk machine group ( Stroke is a major cause of death and global disability . Thirty Previous studies have reported the benefits of overground, treadmill, staircase, and device-assisted gait training on motor and sensory lower limb function, walking endurance, and walking speed in stroke survivors ,10. GamaNeuroplasticity training in hemiparesis after a stroke consists of five principles—specificity, repetition, intensity, time, and salience . Therefon = 15) and I-Walk gait trainer machine group (n = 15) . Block randomization was performed, and the participants were divided into the following two groups: control group , (n = 15) . One dayThirty participants who matched the criteria were recruited from two communities. Inclusion criteria were (1) a first-time stroke with more than 6 months post-stroke, (2) aged between 40 and 70 years, (3) able to walk more than 3 m with or without gait devices, and (4) able to understand or follow commands. Exclusion criteria were (1) unstable cardiovascular problems and (2) musculoskeletal disorders that altered the training program.The I-Walk machine  was designed as a specific tool for assisting walking and controlling walking patterns  with an adjustable number of repetitive walking steps (0–120 steps/min) . Recoil In Conventional Physical Therapy (PT), the control group first received upper limb and hand movement and sit-to-stand training for 30 min followed by overground training for 30 min, 3 days/week for 8 weeks, by a physical therapist.Gait performances were evaluated by the 6-minute walk test (6 MWT) for walking distances and cardiovascular endurance, the 10-meter walk test (10 MWT) for gait speed, and the TUG for detecting dynamic balance during walking at the same points with the primary outcome.The 6 MWT has been used to define and monitor gait performance changes in walking endurance and cardiovascular function to predict community ambulation in stroke survivors. Basic individual demographics are important factors .The 10 MWT has been designed to measure walking or gait speed. It requires a 20-meter, indoor, flat, straight hallway. The first and last 5 m are used for walking acceleration and deceleration, while the time during the middle 10-meter distance is recorded. The participants are asked to walk at a self-selected speed with or without walking aids such as a walker or cane. The speed was calculated by dividing the distance by time (m/s). The test has excellent internal consistency and reliability for individuals with chronic stroke .The TUG, developed by Podsiadlo and Richardson, evaluates functional mobility and dynamic balance while walking . The tesThe secondary outcome of lower limb motor recovery was measured by the FMA at baseline, 2 weeks, 4 weeks, 6 weeks, and at the end of the experiment. The test has high internal consistency and reliability, excellent responsiveness, and high content validity in the population with chronic stroke ,20,21. Dt-test. The significance level was set at 0.05. The sample size was calculated based on a previous study [Data were analyzed using SPSS version 17.0 . All the data are presented as mean ± SD unless otherwise indicated. A two-way mixed-model ANOVA was used to compare all variables within and between groups. Post hoc analysis was conducted using the Bonferroni test, then the statistical approach was tested with a us study .Thirty participants completed gait performance training . All the baselines between both groups were not statistically different, as shown in p < 0.001; effect size (ES) = 0.73), then the statistical approach with the t-test started to increase at 2 weeks (p < 0.05). Meanwhile, the total motor function in both groups significantly increased  = 88.8; p < 0.001; ES = 0.76), while it began to increase at 2 weeks for the I-Walk group (p < 0.05). There was a main time effect in lower limb sensation for both groups in the present study with a repeated measure ANOVA  = 13.9; p < 0.001; ES = 0.33). The conventional PT was significantly higher in lower limb sensation, which began at 2 weeks (p < 0.05), but the I-Walk group started to increase at 4 weeks (p < 0.05). Unexpectedly, the passive joint motion sub-score significantly increased only in the conventional PT group  = 16.9; p < 0.001; ES = 0.38). No significant change was observed for the joint pain sub-score.Six sub-scores of the FMA of lower extremity (FMA-LE) scores of the participants who trained with I-Walk and the overground method are shown in p < 0.05). However, conventional PT tended to gain in passive joint motion compared to I-Walk.In the comparison across groups, no difference was observed in the four lower extremity sub-scores . Two sub-scores (lower extremity and passive joint motion) in the I-Walk group were higher than the conventional PT score at baseline   . There was no group difference in 6 MWT  = 208.4; p = 0.38; ES = 0.03) and TUG  = 107.3; p = 0.36; ES = 0.03). The gait speed of the 10-meter distance in both groups significantly began to change at 4 weeks  = 50.0; p < 0.001; ES = 0.64)  ( = 0.59) A and the = 0.44) B over 8  = 0.64) C. The ga = 0.17) C.The main finding in this study was that I-Walk training improved gait speed more than conventional PT training. However, I-Walk showed slightly decreased passive joint motion. Improvements of lower extremity impairment and gait performances  were found in both conventional PT training and I-Walk training.Based on gait speed, the baseline of the community stroke survivors was between 0.4 and 0.8 m/s . The minThe I-Walk gait training machine sufficiently facilitates locomotor function in chronic stroke in agreement with a previously studied robotic-gait training machine ,27. The Previous studies found that 1000 steps/training session by robotic device training for 4 weeks over a range of levels of weight bearing on the hindlimbs led to motor recovery in animals with ischemic lesions, ,30,31 anGait is a multifactorial task consisting of a healthy cardiovascular system, proper dynamic balance and rhythmic gait speed, and motor control. The 6 MWT is a standard tool widely used for determining walking performance and cardiovascular endurance . CardiovDynamic balance during walking in the community is challenging for individuals with hemiparesis after a stroke . In our The time spent for TUG in our study significantly decreased in both groups compared to the baseline , but it is still inadequate to prevent the risk of a fall based on the fall risk cut-off point, i.e., 14  and 15 sSome machine limitations must be noted. First, it did not give complete walking adaptability experiences, which are important for walking in the community . Second,The I-Walk training machine improves gait speed but slightly decreases the range of motion compared to conventional PT training. I-Walk could maximize patient ability for community ambulators; however, it is suitable for first approach training. Consequently, the various environments that need to be altered challenge the patient’s ability. The conventional PT for the gait rehabilitation might have higher potential by including I-Walk training to improve lower limb impairment. The greater intensity and longer duration of this training should be considered for further implementation in the community.


Background: The cytotoxic effects of COVID-19 raise the question of a possible relation between COVID-19 infection and new-onset type 1 diabetes. We report the case of an eight-year-old boy with new-onset type 1 diabetes and an asymptomatic COVID-19 infection.Case presentation: The eight-year-old boy was hospitalized on December 18th, 2020 due to increased polyuria during the preceding 1 week. Type 1 diabetes was diagnosed with autoantibodies against glutamic acid decarboxylase, tyrosine phosphatase and insulin. The Hba1c value was 11.6%. Upon admission, the PCR test for COVID-19 was positive, the duration of the infection was not clear due to the asymptomatic course, and antibodies were initially negative. Significantly elevated antibodies against COVID-19 were detected 15 days later.Conclusion: The laboratory findings led us to the hypothesis that the boy already had an increased risk of developing autoimmune diseases (HLA DR3-DQB1*02:01 and DR4-DQB1*03:02 positive). The Hba1c value allows speculation that the diabetes manifestation was already “on the way” and that a relatively recent COVID-19 infection could have accelerated the process. The findings are in contrast to a recent report in which COVID-19 infection preceded the manifestation of an insulin-dependent diabetes mellitus by about 5–7 weeks. Due to the paucity of reports, cases with a suspected connection between diabetes mellitus and COVID-19 infection should be entered into the CoviDiab registry (https://covidiab.e-dendrite.com).  During the first two months of the COVID-19 pandemic in Germany, Kamrath et al. . The autth, 2020 because of increasing polyuria during the 1 week prior to admission. The child had lost 1 kg. Ketone bodies (>15 mmol/l) at a pH of 5 and glucosuria of 56 mmol/l were detected in the urine. The glucose value in capillary blood was 20.5 mmol/l (=369 mg/dl). The Hba1c level was 11.6%, corresponding to 103 mmol/mol H (29–42). Since testing for COVID-19 is currently mandatory for new admissions in our clinic, the rapid antigen test was found positive and was then confirmed by a positive PCR. There were no symptoms of an infection of the upper respiratory tract.An eight-year-old boy was hospitalized on December 18th, 2020, the child’s mother noticed a loss of olfaction, which was later accompanied by flu-like symptoms. Her PCR for COVID-19 was also positive.Starting on December 17Interestingly, the child already had a possible history of celiac disease that was diagnosed in a children’s hospital at the age of 2.5 years due to meteorism and diarrhea.  Due to dietary measures, the child was free of symptoms, and surprisingly, the diet could be discontinued at an age of 7 years. Attention deficit hyperactivity disorder (ADHD) was diagnosed a few months ago. There is a family history of diabetes mellitus insofar as a great-aunt had died of diabetes at the age of 18.Autoantibodies relevant for diabetes mellitus type 1: against glutamic acid decarboxylase (GAD): 1172 IU/ml (<10), against tyrosine phosphatase (IA-2-Ab): >4,000 IU/ml (<19), insulin-antibodies 4.81 U/ml (<0.4), against islet cells (IFT): negative, against zinc-transporter 8 (ZnT8-Ab): negative.C-peptide: 0.80 µg/l (0.49–5.52).Human leucocyte antigens: HLA DR3-DQB1*02:01: positive; DR4-DQB1*03:02: positive; DQB1*0:6:02: negative. Assessment: The patient is a carrier of predisposing alleles and has a significantly higher risk of developing type 1 diabetes compared to persons with negative characteristics. The protective allele is negative.Antibodies relevant for celiac disease: the serologic tests for the three antibodies common in celiac disease: anti-tissue transglutaminase (tTG) antibodies, endomysial antibodies (EMA) and deamidated gliadin peptide (DGP) antibodies were negative. IgA: 1.36 g/dl (0.34–3.05). Furthermore, autoantibodies against thyroid antigens were not detected.nd, 2020. On January 3rd, 2021, the PCR for COVID 19 was negative. On January 5th, IgA was 7.11 (<0.8) and IgG was 3.29 (<0.8).Antibody testing against COVID-19 (IgG and IgA) was negative on December 22Course: During the first day, the patient was administered intravenous insulin therapy, on day 2 subcutaneous insulin therapy with an intensified conventional regimen  was initiated. Over the course of the next few days, blood glucose levels stabilized at 8.4–10.3 mmol/l. The patient and his parents received an educational program  according to the guidelines for insulin-dependent diabetes mellitus management. The patient further received a continuous glucose measurement system . He was discharged in a good state, and the total insulin requirement was about 15 IU.Viruses have long been discussed as candidate triggers of islet autoimmunity and type 1 diabetes based on epidemiological and experimental evidence. Enterovirus infection is frequently discussed, but some studies have also reported potential associations of other viruses with type 1 diabetes, including mumps, cytomegalovirus, rotavirus, parechovirus, Epstein-Barr virus, rubella, and parvovirus . The COVIn the first wave of the COVID-19 pandemic, patients were not regularly tested for COVID-19. Thus, some possible coincidences between diabetes manifestation and the infection were not detected in patients who were asymptomatic with regard to COVID-19.Unfortunately, due to the boy's asymptomatic course, it is not possible from biographical data to clarify how long he already had the COVID-19 infection.Looking at the laboratory parameters as a whole, it is possible to initially speculate the following: The boy already had an increased risk of developing autoimmune diseases . The Hba1c value allows speculation that the event was already “on the way” and that a relatively recent COVID-19 infection (see antibody history) could have accelerated the whole process.As for the increased risk for developing autoimmune disease, the association between celiac disease and type 1 diabetes is well known . UnfortuNonetheless, the case report has limitations. The causality between COVID-19 infection and the onset of type 1 diabetes cannot be proven. We know nothing about the speed at which COVID-19 can destroy human beta cells, especially not in a potentially susceptible individual. Thus, next to the possibility that nascent type 1 diabetes mellitus with an acceleration of the process through the cytotoxic effects of COVID-19 occurred, the infection could also have been the primary trigger. The elevated Hba1c does not necessarily mean that hyperglycemia really preceeded COVID-19 infection: variations in red blood cell survival are known that may cause a variation in Hba1c levels for a given mean blood glucose , as the https://covidiab.e-dendrite.com. It is in the nature of casuistic reports not to be able to clarify causality. In addition, they cannot rule out simple coincidence, since COVID-19 infection is frequent in the population. For this reason, the CoviDiab registry was recently put online, in which cases with a suspected connection between diabetes mellitus and COVID-19 infection should be entered: Due to the cytotoxic potential of the COVID-19 virus, negative effects on the pancreatic beta-cells seem possible. In contrast to a recent report, the case described here suggests an acceleration of the problem in an already predisposed individual. To date, there are very few reports; collection and reporting in a registry is mandatory to gain more information about a possible association between COVID-19 infection and insulin-dependent diabetes mellitus.The authors declare that they have no competing interests.There was no financial support.The patient’s parents gave informed consent to the reporting of their child’s case.The authors would like to thank Kerstin Kaiser (diabetes advisor DDG) for the care and training of the child and his parents as well as the grandparents under the extraordinary circumstances described.


Objective: To study the optimal cut-off value of thyroglobulin measurement in a fine-needle aspiration (FNA-Tg) in diagnosing malignant lymph nodes and benign lymph nodes (LNs) according to the thyroid tissue status.Methods: A total of 517 LNs were aspirated: 401 preoperative LNs, 42 LNs after subtotal thyroidectomy and 74 suspected LNs after total thyroidectomy. The cut-off value of FNA-Tg was obtained from receiver operating characteristic (ROC) analysis. The cut-off value with the best diagnostic performance was then obtained by comparing different cut-off values from other studies.Results: LN FNA-Tg levels differed between preoperative and total thyroid disease (p < 0.001) and subtotal thyroidectomy and total thyroidectomy (p = 0.03), but not between preoperative and subtotal thyroidectomy (p = 1.00). Accordingly, those 443 LNs with preoperative and subtotal thyroidectomy were compared to those 74 without thyroid tissue. The optimal cut-off value in thyroid tissue group was 19.4 ng/ml and the area under the ROC curve (AUC) was 0.95 (95% CI 0.92–0.97). The optimal cut-off value in thyroid tissue absence group was 1.2 ng/ml and the AUC was 0.93 (0.85–0.98). After the analysis and comparison of multiple cut-off values, the optimal diagnostic performance was still found to be 19.4 ng/ml and 1.2 ng/ml.Conclusion: The influential factors of FNA-Tg are still controversial, and the optimal cut-off value of FNA-Tg can be determined based on the presence or absence of thyroid tissue. FNA-Tg can be used as an important auxiliary method for diagnosing cervical metastatic LNs of thyroid cancer. Differentiated thyroid cancer includes both papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC), and accounts for ∼70% of all thyroid tissue malignancies , 2. The Thyroglobulin is produced exclusively by follicular cells, and so its expression in nonthyroidal tissue supports the diagnosis of LN metastasis and recurrence of PTC , 14. ManWe broadly hypothesised that the status of thyroid tissue is the main influencing factor, and determined to define the optimal cut-off value of FNA-Tg according to the thyroid status and compare different cut-off values of FNA-Tg to obtain a cut-off value with the best diagnostic performance. The optimal cut-off value of FNA-Tg may can be determined based on the presence or absence of thyroid tissue and FNA-Tg may can be used as an important auxiliary method for diagnosing cervical metastatic LNs of thyroid cancer.Between January 2017 and February 2019, a total of 517 patients (with 517 LNs) with suspicious LNs as detected by ultrasound in the Affiliated Hospital of Qingdao University were collected. The US features suggested metastatic LNs due to the characteristics of microcalcifications (diameter <2 mm), round shape (long diameter to short diameter ratio <2), absence of fatty hilum, cystic changes, calcifications, and peripheral vascularity . All theAll patients underwent ultrasound examination with high-frequency linear-array transducers of 5 to 12 HZ. US-guided FNA was performed using a 22 gauge needle with a 5 ml syringe. The aspirate contents were smeared on slides, stained with Diff-Quik, reviewed for rapid on-site evaluation. The other smears are immediately immersed and fixed in 95% ethanol, and subsequently stained by hematoxylin and eosin in the pathology laboratory. The same residual needle aspirate was washed out in 1.0 ml normal saline for FNA-Tg measurement. The samples were immediately sent to the laboratory and stored at −22°C for 0–4 days for thyroglobulin analysis by an automated electrochemiluminescence immunoassay . The lower level of detection was 0.4 ng/ml, upper level was 500 ng/ml. The positive TgAb was measured with a reference value >115 μ/ml.All FNAC results divided the LNs into positive and negative groups. The non-metastatic LN group included benign, inflammatory, atypical hyperplasia and inadequate cytology. The metastatic LN group included suspected malignant and malignant. The benign and malignant LNs were confirmed by surgical pathological results, which is the gold standard. All patients were tested for FNA-Tg, and only part of the patients measured serum thyroglobulin and TgAb levels.p value < 0.05 was considered significant.SPSS 24.0 software was used for data analysis and image construction. Continuous variables were expressed as mean with standard deviation, and the classified variables were expressed as median (interquartile range). One-way analysis of variance (ANOVA) was performed for the normally distributed variables, non-parametric Kruskal-Wallis test was performed for the non-normally distributed variables. Receiver-operating characteristic analysis (ROC) was performed to determine the optimal diagnostic cutoff value of FNA-Tg in the diagnosis of LN metastasis under different thyroid tissue states. The area under the ROC curve (AUC) was obtained. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy were used to compare the diagnostic value of the different diagnostic methods. Data with a p = 0.84). FNA-Tg and serum thyroglobulin failed to correlate significantly in the thyroid tissue presence group (r = 0.01), with a weak positive correlation in the thyroid tissue absence group (r = 0.21). Seventy-four patients were treated with thyroxine-suppressive therapy in the total thyroidectomy group. The values of FNA-Tg in patients on and off therapy were 57.8 ng/ml (27.2–319.5) and 282 ng/ml (4.4–500.0), respectively (p = 0.74).From 517 patients, 517 LNs were sampled . There wBy constructing ROC curves, the optimal cut-off value for each group was determined according to the highest critical point of the Youden index . The optThe commonly used FNA-Tg cut-off values and different values suggested in other studies were selected, and applied to this study according to different surgical conditions in order to compare the diagnostic performance. In the group with thyroid tissue, the 19.4 ng/ml was the optimal cut-off value . HoweverAs the serum thyroglobulin level was not a routine preoperative measurement, the serum thyroglobulin value was measured in 310 cases in the thyroid tissue group  and 56 cThe measurement of FNA-Tg levels was first proposed by Pacini in 1992, and the mean ±2 times standard deviation of FNA-Tg of benign LN has been suggested to be taken as the best cut-off value for diagnosing with a sensitivity of up to 100% . Some haThere are few other studies that focused on the influence of the presence or absence of thyroid tissue on the cut-off value. Therefore, we mainly focus on the influence of status of the thyroid tissue. Non-parametric Kruskal-Wallis tests were used to confirm statistical difference in FNA-Tg values between the preoperative and the total thyroid tissue groups and between subtotal thyroidectomy and total thyroidectomy groups, while no statistical difference was observed in the FNA-Tg values between the preoperative and subtotal thyroidectomy groups. This indicated that the presence of thyroid tissue has an impact on FNA-Tg values, which was consistent with that of the conclusions put forwarded by Kim and Moon et al. , 26, 27.Based on large scale recruitment of patients in our study, the cut-off values used in this study were those that are commonly used in the past literatures and are used for comparing the diagnostic performance. The commonly used values in the previous studies are FNA-Tg  and FNA-Tg/thyroglobulin ratio >1. In several studies, 1.0 ng/ml is taken as the optimal cut-off value for diagnosing malignant LNs in PTC –17, 20 aHowever, there are certain limitations in our study. First, there was a selection bias, because the patients who underwent FNAC and FNA-Tg had ultrasonic malignant signs. Secondly, there was a difference in the number of patients between the group with thyroid tissue and the group without thyroid tissue, and the number of cases collected in the two groups was not balanced in the same period. Therefore, it is necessary to further expand the sample number of patients after total thyroidectomy to reduce the interference of various uncertain factors.Our data represent an advance in biomedical science in that it demonstrates a method for determining the likelihood of the malignant invasion of lymph nodes in thyroid cancer.• Thyroglobulin measurement in the washout fluid of the needle used for FNAC aspiration (FNA-Tg) is a useful tool for improving the diagnostic accuracy of FNA.• However, the optimal cut-off value of FNA-Tg is uncertain and the results of current studies are controversial.• FNA-Tg of 19.4 ng/ml and 1.2 ng/ml can be used as optimal cut-off values for the presence or absence of thyroid tissue, respectively.• FNA-Tg can used as an important auxiliary method for diagnosing cervical metastatic LNs of thyroid cancer.


We report a 30-day mortality of 2.8% in the S3 group, and 2.1% in the ER group (p = 0.674). There was no difference in stroke, conversion to open surgery, vascular and bleeding complications or myocardial infarction. While prosthesis mean gradients were higher with S3 , there was a trend to less paravalvular regurgitation . All-cause mortality up to 5 years did not show a difference . Independent predictors of long-term mortality were impaired LVEF, chronic kidney injury, peripheral artery disease, malignant tumor and periprocedural stroke. New generation TAVR valves offer an excellent implant and outcome success rate. Long-term survival was independent of prostheses choice and mainly attributed to comorbidities and complications.The aim of this study was to compare patients with transcatheter aortic valve replacement (TAVR) receiving new generation prostheses SAPIEN 3  and Evolut R  in terms of periprocedural and long-term outcome. Our retrospective, single-center analysis included 359 consecutive patients with severe aortic stenosis who underwent TAVR with S3 or ER from 2014–2016 . Device Success was equal (S3 93.0% vs. ER 92.4%,  Since the first transcatheter aortic valve replacement (TAVR) in 2002  percutanBetween June 2014 and May 2016, 489 consecutive patients underwent TAVR in the Heart Valve Center Mainz, Germany. We included 359 patients with severe degenerative aortic valve stenosis treated via transfemoral access using a new generation prosthesis (S3 or ER) in the study. Exclusion criteria were transapical access, valve-in-valve-procedure or the use of a non-ER or -S3 prosthesis type . The assPatient characteristics, imaging and procedural parameters were retrospectively recorded in a Microsoft Excel database . The peri- and post-procedural outcomes of these patients were analyzed according to the updated criteria of the Valve Academic Research Consortium (VARC-2) . In addiA follow-up on mortality status until August 2019 could be achieved in 92% of the patients through state civil registries and our University outpatient department. The study was approved by the local ethics committee (number 2019-14692).t-test for nominal and metrical variables, respectively, if normally distributed, and with Mann-Whitney-U-Test if not normally distributed. Kaplan-Meier survival analysis was used for time dependent all-cause mortality. Comparison of the different groups was carried out with Cox proportional hazard models and Log-rank test. All analyses were performed with IBM SPSS Statistics version 24  and GraphPad PRISM version 8 .All data is described as absolute numbers and percentage in case of nominal variables, as well as mean with standard deviation in case of metrical variables. Differences between patient groups were tested by Chi-squared test and Student’s The study population consisted of 359 patients with severe aortic stenosis who underwent transfemoral TAVR with a new generation prosthesis. The median follow-up period was 3.8 years . The SAPIEN 3 group included 215 patients, the Evolut R group 144 patients.p < 0.001), had a history of myocardial infarction  and were suffering from hypertension . S3 patients also had a lower mean left ventricular ejection fraction . In the ER group the prevalence of diabetes  and pulmonary hypertension  was higher, additionally these patients had a smaller calculated aortic valve area .Baseline patient characteristics are displayed in p = 0.751), but need for post-dilatation was substantially higher in ER patients . Fluoroscopy time and volume of contrast did not differ between both groups.Choice of prosthesis size differed significantly between both groups . Whereasp = 0.812). We report a 30-day mortality of 2.8% in the S3 group and 2.1% in the ER group, respectively (p = 0.674). There was no difference in stroke rate, conversion to open heart surgery, major vascular complications, life-threatening or disabling bleeding or myocardial infarction. The VARC-2 composite endpoint of Early Safety at 30 days also did not differ significantly between both groups . Patients in the S3 group had a higher incidence of periprocedural acute kidney injury which did not reach statistical significance Peri- and post-procedural outcome parameters are shown in p = 0.002). While prosthesis mean gradients were higher in the S3 group , there was a tendency to less paravalvular leaks . Furthermore, there was early improvement of Left Ventricular Ejection Fraction, Mitral Regurgitation and Systolic Pulmonary Pressures at 30 days, which showed consistent values at one year follow up (Implantation of a new permanent pacemaker (PPI) was lower in the S3 group . Subgroup analysis demonstrated a survival benefit for S3 patients with peripheral disease  or history of stroke . Additionally, male patients seemed to benefit from S3  and female patients from ER implantation , although both did not reach statistical significance , peripheral artery disease , chronic kidney injury  and history of cancer . In multivariate analysis (p = 0.005), peripheral artery disease , chronic kidney injury , COPD , history of cancer  and periprocedural stroke . However, the type of prosthesis was no predictor of mortality .Univariate analysis  showed aanalysis  independWith this study we present a comparison of the new-generation aortic valve prostheses Edwards SAPIEN 3 and Medtronic Evolut R concerning long-term as well as peri- and post-procedural outcomes. The analyzed cohort consisted of patients at intermediate to high surgical risk expressed through a EuroSCORE II of 8.0% and STS Score of 7.3% in mean, respectively. Device success in both groups was comparably good and is in line with previous studies using VARC-2 definitions ,11. AlthRegarding periprocedural outcomes, both prostheses showed similar favorable results with very low incidences of stroke, vascular or bleeding complications as well as myocardial infarction or conversion to open heart surgery. Nevertheless, acute kidney injury stage 2 or higher was tendentially more often recorded in the S3 group. Finkelstein et al. describe similar observations, while other studies do not see differences between both prostheses ,13,14. Ap = 0.088) which can be related to the newly developed outer skirt on the SAPIEN 3 prosthesis. Another confounder could be a selection tendency towards ER in very calcified annuli/LVOT that would lead to worse sealing. A further development of the Evolut platform with an added sealing skirt was not available at the time of patient inclusion .In general, we could show a very low incidence of relevant paravalvular regurgitation (moderate or severe PVL 1.9%), a complication that has been associated with worse long-term survival before . These fThe numbers of postinterventional need for permanent pacemaker seem high in comparison to similar studies , which could be explained by a low threshold for implantation in our institution at that time (including persisting left bundle branch block with AV-Block °I or bradyarrhythmia). However, the difference between both groups in favor of SAPIEN 3 (ER patients had 1.6-fold higher PPI rates compared to S3 patients) is comparable to the literature ,19,20.Despite treating a population at substantially increased risk, the 30-day mortality was very low at 2.5% overall with no relevant difference between both groups. Previous studies show comparable mortality data  with transfemoral TAVR patients at lower to similar risk profile  ,18,19,20p = 0.732). A recent work by Finkelstein et al. presented data with the same prostheses in low-risk patients showing similar mortality between both groups up to 3 years [p = 0.15) [Regarding survival there was no difference between S3 and ER patients with a mean survival of 3.5 and 3.3 years, respectively. This finding could also be confirmed in multivariate analysis  .As in most studies concerning TAVR, long-term survival was mainly attributed to comorbidities such as reduced LVEF, peripheral artery disease, chronic kidney injury, advanced COPD or history of cancer. In addition, periprocedural stroke was the most important independent predictor of all-cause mortality up to five years. This finding is supported by a work from Levi et al. that showed a similar relevance of in-hospital stroke for long-term survival .The present study has to be interpreted with regard to several limitations. First, it is a retrospective analysis that has inherent limitations and is open to bias. Second, the study was conducted at a single center, thus results might not be generally applicable. Third, the choice of the platform was not randomized but up to the decision of the implanter which could result in biased periprocedural outcome parameters. Since 34 mm ER was not on the market in the reported inclusion time, there might be a bias towards treating more patients with larger annuli with the S3 29 mm valve, which could explain the higher number of male patients treated in the SAPIEN group. At 5 years after implantation the number of patients that are included in the follow-up is low, thus the median follow-up time of 3.8 years could also be addressed as intermediate- to long-term follow-up.Transcatheter aortic valve replacement (TAVR) has become a standard therapy in treating AS. Published data regarding long-term outcomes in patients with new generation TAVR prostheses  are scarce.We therefore present a large single center analysis of TAVR with SAPIEN 3 and Evolut R valves in comparison with focus on long-term follow up. Our single-center cohort data adds an industry-independent comparison between new-generation valves concerning periprocedural outcomes and long-term survival in a routine TAVR cohort. We demonstrated low procedural mortality and low paravalvular leakage rates in new generation TAVR. Mid- to long-term follow-up shows favorable similar survival in both prosthesis groups.


Objective: Immune checkpoints inhibitors are promising and wide-spread agents in anti-cancer therapy. However, despite their efficacy, these agents could cause cardiotoxicity, a rare but life-threatening event. In addition, there are still no well-described predictive factors for the development of immune-related adverse events and information on high risk groups. According to known experimental studies we hypothesized that cardiovascular diseases may increase myocardial PD-L1 expression, which could be an extra target for Checkpoint inhibitors and a potential basis for complications development.Methods: We studied patterns of myocardial PD-L1 expression in non-cancer-related cardiovascular diseases, particularly ischemic heart disease (n = 12) and dilated cardiomyopathy (n = 7), compared to patients without known cardiovascular diseases (n = 10) using mouse monoclonal anti-PD-L1 antibody . Correlation between immunohistochemical data and echocardiographic parameters was assessed. Statistical analyses were performed using R Statistical Software—R studio version 1.3.1093.Results: In the myocardium of cardiac patients, we found membranous, cytoplasmic, and endothelial expression of PD-L1 compared to control group. In samples from patients with a history of myocardial infarction, PD-L1 membrane and endothelial expression was more prominent and frequent, and cytoplasmic and intercalated discs staining was more localized. In contrast, samples from patients with dilated cardiomyopathy displayed very faint endothelial staining, negative membrane staining, and more diffuse PD-L1 expression in the cytoplasm and intercalated discs. In samples from the non-cardiac patients, no convincing PD-L1 expression was observed. Moreover, we discovered a significant negative correlation between PD-L1 expression level and left ventricular ejection fraction and a positive correlation between PD-L1 expression level and left ventricular end-diastolic volume.Conclusions: The present findings lay the groundwork for future experimental and clinical studies of the role of the PD-1/PD-L1 pathway in cardiovascular diseases. Further studies are required to find patients at potentially high risk of cardiovascular adverse events associated with immune checkpoint inhibitors therapy. Programmed cell death receptor 1 (PD-1) and its ligand PD-L1 are involved in the regulation of T-cell activation, tolerance, and immune-mediated organ damage. Under physiological conditions, PD-1/PD-L1 signaling plays an important role in the prevention of autoimmune diseases. Apart from the expression on T- and B-cells, dendritic cells, and macrophages, PD-L1 could be expressed on non-hematopoietic cells, including cardiomyocytes and endothelial cells. Recently, it was found that a wide range of tumor cells express PD-L1 on their surface to prevent antitumor immune response (Therapy with immune checkpoint inhibitors (ICI) was associated with increased overall survival in patients with advanced cancer that previously had a poor prognosis. The results of KEYNOTE-042 trial comparing the effectiveness of ICI and standard chemotherapy in patients with advanced or metastatic non-small cell lung cancer (NSCLC) and PD-L1 tumor proliferation score (TPS) >50% showed that relapse-free survival was better in a group of ICI therapy−20 months vs. 12.2 months in the standard chemotherapy group . The lonCardiotoxic side-effects of ICI therapy have been reported since 2016. First publications described the development of fulminant myocarditis in patients receiving ICI –7. MoreoIn vivo experiments performed by Grabie et al. discovered that IFN-γ-induced PD-L1 was mainly expressed on endothelium and its expression had an important cardioprotective effect against immune-related heart damage  disease and traditional CV risk factors in cardiac irAEs occurrence is not yet fully understood. Histological and immunohistochemical analysis revealed high levels of membrane and cytoplasmic PD-L1 expression in samples from patients with ICI-associated myocarditis , 16. Howt damage . Later, t damage . Baban et damage .Up-regulation of PD-L1 may probably attenuate T-cell response against damaged cardiomyocytes, for example, in the course of ischemic heart disease (IHD), thus reducing the local inflammation in the myocardium. On the other hand, high PD-L1 myocardial expression in CV diseases might be associated with an increased risk of developing irAEs, since PD-L1 is a direct target for anti-PD-1 and anti-PD-L1 ICI. However, there is still no research demonstrating increased myocardial expression of PD-L1 in damaged human hearts due to different types of CV diseases.To characterize the PD-L1 expression pattern in patients with CV diseases of different etiology, we analyzed PD-L1 myocardial expression in patients with documented IHD and dilated cardiomyopathy (DCM).+ T-cells and CD68+ macrophages. Additionally, the correlation analysis between echocardiographic parameters, complete blood count and histological results has been conducted (We examined 12 autopsy samples of left ventricular (LV) myocardium obtained from patients with a history of myocardial infarction (MI). Nine patients died in an acute period of MI. Cardiac pathology specimens from an infarct-related artery were used for further immunohistochemical evaluation. The comparison group included seven samples of LV from patients with DCM who underwent orthotopic heart transplantation. In this group, IHD was excluded according to coronary angiography results. Echocardiography was carried out for all patients at one clinic. The control group included 10 LV samples collected from cancer patients without known CV pathology who died in the early postoperative period and had not received neoadjuvant chemotherapy and/or immunotherapy and 4 LV samples from patients received ICI and died without intravital data for CV irAEs (two without CV diseases and two with known IHD). The tissue was fixed in 10% neutral buffered formaldehyde and then embedded in paraffin. We used hematoxylin and eosin (HE) staining to visualize the myocardial structure and immunohistochemistry to investigate the expression of PD-L1 and distribution of CD3onducted .The study was approved by the local ethics committee .Immunohistochemistry was performed on the automated immunostaining platform Autostainer Link 48  for PD-L1 and Ventana Benchmark Ultra  for CD3. Tissue sections were immunostained with mouse monoclonal anti-PD-L1 antibody , rabbit monoclonal anti-CD3 antibody  and mouse monoclonal anti-CD68 antibody . All slides were scanned using a Pannoramic 1000 scanning microscope (3D Histech) with a x60 objective lens. Assessment of PD-L1 was performed by an experienced board pathologist. CD3 and CD68 expression was quantify with digital image analysis of scanned by QuPath software. We assessed membrane, cytoplasmic, and endothelial PD-L1 expression in all groups.To characterize the expression level of PD-L1 in the myocardium, we developed a combined cardiomyocyte positive score (CMPS). CMPS was calculated as a percentage of PD-L1 positively stained cardiomyocytes with membrane and/or cytoplasmic expression of any intensity. To additionally evaluate the PD-L1 expression in intercalated discs (ICD), we determined the PD-L1 ICD positive score (ICDPS), which was defined as a percentage of positively stained ICD from all cardiomyocytes cut in a longitudinal section.p-values <0.05 were considered significant. All statistical analyses were performed using R Statistical Software—R studio version 1.3.1093.Data were expressed as mean and standard deviation (Mean ± SD) or median with 25th and 75th percentiles . Clinical and expression data were analyzed using the Mann-Whitney U test for continuous variables and Fisher Exact test for dichotomous variables. Correlations were calculated with Spearman's rank correlation coefficient for non-parametric samples. n = 9), the mean time interval between symptom onset and death was 7.9 ± 4.3 days. During hospitalization, 10 of 12 patients underwent percutaneous transluminal coronary angioplasty, and one patient underwent coronary artery bypass graft surgery. The mean age in the DCM group at the time of heart transplantation was 52.1 ± 9.8 years, which was significantly lower than in the MI group (p = 0.008). There were no age differences between the experimental and control groups. Control group age was 59.5 ± 12.4 (p = 0.197 for MI and p = 0.186 for DCM). Clinical, echocardiography, and immunohistochemistry data of studied groups (MI and DCM) are summarized in The mean age in the MI group at the time of death was 66.1 ± 7.0 years. For patients who died from acute MI  cells per mm2], so inflammation cardiomyopathy was excluded . In contrast, PD-L1 expression was not detected in patients without CVDs in ICI therapy group  and ICDPS  compared to the MI group.There were no statistically significant differences between experimental groups in the presence or absence of different expression patterns. However, DCM group had significantly higher CMPS  and between ICDPS and LVEF , and significant positive correlations between CMPS and LVEDV  and between ICDPS and LVEDV . After dividing patients into subgroups, a significant negative correlation between ICDPS and LVEF  remained only in the DCM group. In the group of MI, only the tendency to a positive correlation between ICDPS and LVEDV was found . The lack of other significant correlations between studied parameters in subgroups may be attributed to a low number of analyzed samples.According to correlation analysis between immunohistochemistry and echocardiography data, we got the following results . In all Also, we have indicated strong positive correlations between myocardial PD-L1 expression  and complete blood count  for patients with IHD . No corrThe present study aimed to examine the expression profiles of PD-L1 in the myocardium of cardiac patients without a history of cancer. In damaged myocardium from patients with IHD and DCM, we found several patterns of PD-L1 expression compared to the myocardium of patients without CV diseases. The presence of membrane and endothelial expression was more specific for patients with MI history than those without ischemic damage. The reason for this result is not yet fully understood. Previously, PD-L1 endothelial expression was described in the mouse model of CD8 T-cell myocarditis . TherefoFurther analysis revealed PD-L1 expression in intercalated discs in all groups of cardiac patients, but predominantly PD-L1 was observed in the DCM group. This matches well with the recently described strong but diffuse staining of PD-L1 in ICD of cardiac allograft vasculopathy hearts. However, the staining was considered by the authors as non-specific and insignificant .p = 0.01). The limitation of our study is that we used archived material presented by paraffin blocks which makes it impossible to conduct flow cytometry or ELISA assay to describe T-cell immunophenotyping and cytokines levels.The crucial role in ICI toxicity development plays activation of inflammatory response which damage tissues and organs. Cytokines that are secreted by immune cells such as macrophages, activated T-cells, B-cells and NK cells take a lead in irAEs pathophysiology. Increased levels of IFN-γ and IFN-γ pathway genes are positive biomarkers of tumor response on ICI treatment and irAEs and IL-8, IL-6, and TGF-β are negative biomarkers . ExperimIn large retrospective study Oren et al. showed the increasing of ICI-related myocarditis risk from 0.13% to 4.5% in patients with history of MI, HF and age >80 years . But theTo sum up, our work described an increase of PD-L1 expression in the myocardium of cardiac patients and revealed a correlation between PD-L1 expression  and echocardiographic parameters of left ventricular size and function (LVEDV and LVEF). The findings of this study lay the groundwork for further investigations aimed to identify the high risk patients for CV irAEs and give us a reason to pay more attention to patients with LV dysfunction and heart chambers enlargement.We are aware that our research may have the limitation of a small sample size that did not allow us to investigate additional correlations in distinct subgroups of cardiac patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved by the local ethics committee . The patients/participants provided their written informed consent to participate in this study.EK performed data collection, statistical analyses, and wrote manuscript. VK performed immunohistochemical analysis. AA, LM, and OM provided the critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This study has been supported by the grant from the Ministry of Science and Higher Education of the Russian Federation (Agreement No. 075-15-2020-901).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study investigated temporal trends in the treatment and mortality of patients with cardiogenic shock (CS) in Taiwan in relation to acute myocardial infarction (AMI) accreditation implemented in 2009 and the unavailability of percutaneous ventricular assist devices.Data of patients diagnosed as having CS between January 2003 and December 2017 were collected from Taiwan’s National Health Insurance Research Database. Each case was followed from the date of emergency department arrival or hospital admission for the first incident associated with a CS diagnosis up to a 1-year interval. Measurements included demographics, comorbidities, treatment, mortality, and medical costs. Using an interrupted time-series (ITS) design with multi-level mixed-effects logistic regression model, we assessed the impact of AMI accreditation implementation on the mortality of patients with AMI and CS overall and stratified by the hospital levels.5 person-years increased from 17 in 2003 to 25 in 2010 and plateaued thereafter. Average inpatient costs increased from 159 125 points in 2003 to 240 993 points in 2017, indicating a 1.5-fold increase. The intra-aortic balloon pump application rate was approximately 22–25% after 2010 (p = 0.093). Overall, in-hospital, 30-day, and 1-year mortality declined from 60.3%, 63.0%, and 69.3% in 2003 to 47.9%, 50.8% and 59.8% in 2017, respectively. The decline in mortality was more apparent in patients with AMI-CS than in patients with non-AMI-CS. The ITS estimation revealed a 2% lower in-hospital mortality in patients with AMI-CS treated in district hospitals after the AMI accreditation had been implemented for 2 years.In total, 64 049 patients with CS  were identified. The incidence rate per 10In Taiwan, the burden of CS has consistently increased due to high patient complexity, advanced therapies, and stable incidence. Mortality declined over time, particularly in patients with AMI-CS, which may be attributable to advancements in AMI therapies and this quality-improving policy.The online version contains supplementary material available at 10.1186/s13054-021-03820-1. The clinical features of cardiogenic shock (CS) have considerably changed over the past two decades because of improved knowledge regarding its pathophysiological mechanisms, therapeutic advancements, and collaborative care –6. In-hoThe government has implemented accreditation program for AMI to improve quality of care since 2009 in Taiwan. The impact of accreditation program on outcomes in CS patients has not been evaluated. Since percutaneous VADs including Impella  and TandemHeart  are not available in Taiwan, the impact of inaccessibility of such devices on clinical outcomes is not known. Therefore, we conducted a nationwide longitudinal cohort study by using real-world data to investigate temporal trends in the incidence, medical costs, mechanical circulatory support (MCS) devices, and mortality of CS in Taiwan over the past 15 years. The findings of this study can improve our understanding regarding the features of CS with heterogenous etiologies.International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) and International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) since 2016. The NHIRD can be linked with the National Death Registry (NDR) by using the unique encrypted identification number of each beneficiary. The accuracy of data in the NHIRD and NDR has been analyzed in previous studies [The universal compulsory National Health Insurance (NHI) program was launched by the Taiwanese government in March 1995. Taiwan’s National Health Insurance Research Database (NHIRD) contains complete information regarding outpatient and emergency visits, hospital admissions, medication prescriptions, disease diagnoses, medical procedures, and vital statuses for 99% of Taiwan’s population (~ 23 million). Diagnoses in the NHIRD are coded according to the  studies , 12. TheTo examine the effect of AMI accreditation on the mortality of patients with AMI-CS, we adopted an interrupted time-series (ITS) study design. In brief, ITS is a robust quasi-experimental design in which randomized controlled trials cannot be used. In an ITS design, data are collected at multiple and equally spaced time points  before and after intervention . The main objective of an ITS is to examine changes in the data pattern pre- and post-intervention . In thisn = 213), (2) were younger than 18 years (n = 687), and (3) had a length of stay of more than 365 days (n = 14). The last exclusion criterion was added because primary endpoints could not be evaluated. Each case was followed from the date of emergency department arrival or hospital admission for the patients’ first incident associated with a CS diagnosis up to a 1-year interval.From the NHIRD, we identified patients who received a diagnosis of CS (ICD-9-CM code 785.51 or ICD-10-CM code R570) in an inpatient or emergency department between January 1, 2003, and December 31, 2017. We excluded patients who (1) had missing age or sex information , are recorded and reviewed by the central or local health authorities for the level-specific quality assessment. For example, at least 80% of patients with STEMI must receive initial electrocardiography within 10 min, dual-antiplatelet and fibrinolytic therapies within 30 min, or percutaneous coronary intervention (PCI) within 90 min as required in severe-level hospitals . MoreoveOutcomes were defined as short-term  and long-term (1-year) mortality; these data were obtained from either the NHIRD or NDR. Patients were considered to have died in the hospital if their death record was issued in the hospital or within 2 days after the date of hospital discharge.The selected covariates in the study were thought fundamentally or prognostically important for patients with CS, namely hypertension, dyslipidemia, coronary artery disease, prior myocardial infarction, renal failure, congestive heart failure, peripheral arterial disease, and atrial fibrillation , 7–10. Pp  < 0.05 was considered statistically significant.We presented individual-level data for demographics, incidence, healthcare resource use, medical costs, and mortality of patients with CS across different time periods. The chi-squared test for linear trends was employed to examine changes of baseline over time. A multi-level mixed-effects logistic regression model that has a random intercept for center/hospital of care was used to examine the policy effect on in-hospital mortality before (2003–2008) and after 2009–2017) the introduction of AMI accreditation . Also, w the intr5 person-years increased from 17 in 2003 to 25 in 2010 and plateaued thereafter, with annual numbers ranging from 4500 to 5100. In addition, average inpatient costs increased from 159 125 points in 2003 to 240 993 points in 2017, approximately US$5304 in 2003 to US$8033 in 2017 . Approximately 10% of patients with CS received ECMO therapy, surgical but not percutaneous VADs were implemented sporadically, with approximately 20 cases per year reported after 2012. Ultimately, only 0.3% of patients with CS received heart transplantation. Baseline data stratified by AMI etiology and 1-year mortality are also presented in Additional files Table The unadjusted data showed that in-hospital, 30-day, and 1-year mortality declined from 60.3%, 63%, and 69.3% to 47.9%, 50.8%, and 59.8%, respectively, gradually decreasing over the entire study period Fig. . Moreovep = 0.171). In terms of the effect of AMI accreditation involving longer periods of time, the association of the policy with mortality remained nonsignificant. When analyses were stratified by the hospital level, a 2.0% lower in mortality  was observed in patients treated in district hospitals after the AMI accreditation had been implemented for 2 years. Moreover, the effect continued into later years .The ITS estimation  in our study. Healthcare authorities must draw more attention to the treatment of non-AMI-CS and prevent medical futility in AMI-CS treatment.Our study has several limitations. First, the current study was an observational retrospective claim-based study in the real-world settings. Because clinically relevant imaging and laboratory data were unavailable, the validity of CS diagnosis was mainly dependent on discharge claims. Second, eliminating disease heterogeneity  in our study population was difficult and may have affected our finding in relation to the effect of AMI accreditation. Third, we were unable to study medications and devices not covered by the NHI program . Fourth, we recognized that the impact of AMI accreditation would be weakened since we were not able to directly compare the mortality of patients treated or not treated in accredited hospitals. Finally, other AMI-relevant interventions might affect the in-hospital mortality of AMI-CS patients. Since none of those interventions were implemented nationwide, the impact was likely to be small.The burden of CS has consistently increased in Taiwan because of high patient complexity, advanced therapies, and stable incidence. Without available percutaneous VADs, IABP utilization rate remained unchanged (20–24%). The in-hospital mortality rate decreased from 60% in 2003 to < 50% between 2015 and 2017, but 1-year mortality remained high (60%). In particular, a lower mortality risk was observed in the AMI-CS group than that in the non-AMI-CS group after 2009, possibly reflecting advancements in AMI therapies and the quality-improving policy. Promotion of the post-acute care in patients with CS and therapeutic interventions focusing on patients with non-AMI-CS are essential.Additional file 1. Annual hospital number according to their accredited level for emergency medical ability.Additional file 2. Disease diagnostic coding, procedure coding, and ATC code for medication.Additional file 3. Healthcare resource use stratified by different time periods.Additional file 4. Annual trends of mechanicals circulatory support devices.Additional file 5. Baseline characteristics of patients with cardiogenic shock stratified by AMI etiology.Additional file 6. Baseline characteristics of patients with cardiogenic shock stratified by 1-year mortality.